Abstract

Telomere maintenance in cancer cells can be dis-regulated by introducing small organic molecules, capable of stabilizing the 3\#8217; single-stranded overhang of telomeric DNA as G-quadruplex structures and competing for telomerase and hPOT1 capping proteins. These quadruplex are characterized by guanine-guanine interactions, rather than classic Watson-Crick base pairings. The G-quadruplex basic structural motif is a G-tetrad, a planar surface where four guanines interact by Hoogsteen hydrogen bonds. When two or more G quartets stack together by \#960;-\#960; interactions, the result is a quadruplex structure characterized by a hydrophobic core and four grooves, connected by phosphodiester backbones. Many different G-quadruplex ligands have been synthesized, but the specificity and the activity in vitro/in vivo of the majority has not been sufficient for them to progress further. We have focused on an approach for telomere targeting drug discovery that uses the synthesis of libraries derived from the combinations of different building blocks. A shortcut to obtain a library is the use of reactions that have products that are easy to purify, are efficient and that rarely give rise to side products. \#8220;Click-chemistry\#8221; azide-alkyne Huisgen cycloaddition is an attractive approach that fulfils these objectives. We have synthesized two classes of building blocks, azides and alkynes, which include an aromatic core and a basic side chain that is positively charged under physiological conditions. With these fragments, we have generated a library of G-quadruplex ligands, extending the aromatic surface by the Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition, that gives rise to 1,4-triazole ring formation. The aromatic core and triazole rings in the final compounds are appropriate for interactions with the G-quartets in a quadruplex, and two positively charged side chains interact with the grooves. The affinity and selectivity of these compounds for G-quadruplexes has been evaluated by binding experiments using a FRET melting method. Their biological activity has been studied by SRB assays using two different cancer cell lines that express telomerase, MCF7 and A549, as well as normal fibroblast cell line, WI38. The long-term growth arrest properties of the most active compounds have been evaluated, as well as their activity in combination with cis-platinum. At the same time, a new class of potential G-quadruplex ligands has been designed that are based on these click compounds, in order to investigate the influence that a third side chain can have on G-quadruplex stabilization and specificity. Molecular modeling studies have predicted that such compounds will have significantly enhanced quadruplex affinity and low or no affinity for duplex DNA. Several core groups have being functionalized with different side chains and will be reported at the meeting.